rna silencing, mechanism and applications · rna silencing, mechanism and applications 2. the role...
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RNA Silencing, mechanism and applications
The Central Dogma of Molecular Biology
There is “life” outside protein-coding genes
• There are approximately 70.000 gene predictions for the Human genome
…But the estimated number of protein coding genes is 21,561
• It is estimated that 98% of the transcriptional output of the human genome represents RNA that does not encode protein
RNA Silencing, mechanism and applications
2. The role of small RNAs in gene regulation
3. Mechanisms of RNA interference
4. Biological role of RNA silencing
5. Applications of RNA silencing
6. miRNAs
1. History and definitions
Posttranscriptional gene silencing (PTGS)Posttranscriptional gene silencing (PTGS)
Classical key experiment in petunia:
• Artificially introduced 2 nd CHS gene
• Expected: more intense flowers
Real outcome: Variable phenotypes, including white flowers
• Key enzyme for flower pigmentation:
chalcone synthase (CHS)
van der Krol et al., Plant Cell 2:291 (1990) + Plant Mol Biol 14:457 (1990).
Effects of expression of CHS sense and antisense RNA on flower pigmentation in Petunia
•The silencing of an endogenous
gene due to the presence of a
homologous transgene or virus.
•Co-suppression can occur at the
transcriptional or posttranscriptional
level.
CO-SUPPRESSION
Key experiment on RNA interference (RNAi)
Nature 391, 806 - 811 (1998);Nature 391, 806 - 811 (1998);
Injection of ds RNA to C. elegans causes
gene-specific silencing
Injection of ds RNA to C. elegans causes
gene-specific silencing
New term:
RNA interference (RNAi)
New term:
RNA interference RNA interference (RNAi)(RNAi)
Entrez PubMed Search Terms: RNAi or siRNA or miRNA
Number of Publications
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23.000
RNA silencing
• RNA mediated impairment of transcription- TGS-methylation (shRNAs, rasiRNAs and siRNAs?)
• RNA mediated impairment of translation-PTGS (siRNAs and some miRNAs) and -translational arrest (miRNAs)
Small RNA classification
small RNAs
Sequence specific RNAdegradation
Post-Transcriptinalgene silencing
Short interfering
RNA (siRNA)
Inhibition of translation
Gene regulation
microRNA miRNA
RNA mediated methylation
rasiRNAs, endo-siRNAs etc.
Regulation of the Epigenome
Various types of suppression
Suppression of transposable elements
stress response etc
piRNAtasiRNA
natsiRNA
2. The role of small RNAs in gene regulation
1. History
RNA σίγηση, ένας μηχανισμόςρύθμισης και άμυνας
short interfering RNA (siRNA)short interfering RNA (siRNA)
NN 5’
5’
ca 21 NucleotidesNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNN
3’
3’
short interfering RNA (siRNA)short interfering RNA (siRNA)
NNca 21 Nucleotides
NNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNN
3’
3’
- N N N
- N
N -
N N N -two 3’ protruding
unpaired nucleotides
5’ P -- P 5’
5’ terminal phosphates
PTGS / RNAi inductionPTGS / RNAi induction
Target RNA
antisense RNA
RNA quant( threshold )
“aberrant”RNA
Ectopic
pairing
ds
RNARdRP?
RdRP? ?
Basic Model for the MechanismBasic Model for the Mechanism
Target RNA
antisense RNA
RNaseRNase IIIIII--type enzyme type enzyme DicerDicer
Target RNA
antisense RNA
RNaseRNase IIIIII--type enzyme type enzyme DicerDicer
Basic Model Basic Model
short short interfering interfering
RNARNA( siRNA )( siRNA )
Basic Model Basic Model
short interfering RNAshort interfering RNA
siRNAs play an essential role in RNA interference
They are mediators as well as indicators of posttranscriptional gene silencing (PTGS)
Integration of siRNA to RISCIntegration of siRNA to RISC
N
N
N
N
N
N
N
N
N
N
N
N - P 5’
P -5’
ca 21 Nucleotides
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Incorporation in
RNA-induced silencing complex
(RISC complex)
Degradation of Target RNADegradation of Target RNA
targetRNAs
cleavedtargetRNAs
Degradation of target RNA: major playersDegradation of target RNA: major players
DicerDicer RISCRISC
siRNAsiRNA
Dicer
• Dicer generates RNAs with 2 nt 3’overhang and 5’ phosphorylated terminus, both required for activity
• Fly Dicer requires ATP, human may not
Bact.RNase III
Class II: DroshaClass III dicer
ENZYME STRUCTURE – RNase III
Different organisms have different numbers of Dicers
Mello CC, Conte D; Nature 431:338, 2004
Transgenic GFP expressing N.benthamiana is our system
Silencing in plants is a non-cell autonomous event
The Mechanism of RNA silencing is to a certain extent conserved
between eucaryots
Testing plant extracts for their silencing potentialTesting plant extracts for their silencing potential
mRNA
si RNAGFP non-silenced GFP silenced
RNA extract
GFP-expressing
C.elegansRNA extract
RNA silencing pathways in fungi
RNA silencing pathways in fungi
AS, ascomycete; BS, basidiomycetes; ZY, zygomycete; OO, oomycete; AC, Aspergillus clavatus; AFl, Aspergillusflavus; AFu, Aspergillus fumigatus; AN, Aspergillus nidulans; AO, Aspergillus oryzae; AN, Aspergillus terreus; CA, Candida albicans; CC, Coprinus cinereus; CL, Candida lusitaniae; CN, Cryptococcus neoformans; CT, Candidatropicalis; FG, Fusarium graminearum; FO, Fusarium oxysporum; FV, Fusarium verticillioides; HC, Histoplasmacapsulatum; MO, Magnaporthe oryzae; NC, Neurospora crassa; PC, Phanerochaete chrysosporium; PG, Pucciniagraminis; PI, Phytophthora infestans; RO, Rhizopus oryzae; SC, Saccharomyces cerevisiae; SN, Stagonosporanodorum; SP, Schizosaccharomyces pombe; UM, Ustilago maydis; AT, Arabidopsis thaliana.
In fungi there seems to be an avoidance of RNAi when “killer”
virus is present
2. The role of small RNAs in gene regulation
3. Mechanisms of RNA interference
4. Biological role of RNA silencing
1. History and definitions
RNA σίγηση, ένας μηχανισμόςρύθμισης και άμυνας
Biological role of silencing
Protection against invasive RNAsespecially RNA viruses
Silencing of transposable elements
Regulation of expression of endogenous genes
2. The role of small RNAs in gene regulation
3. Mechanisms of RNA interference
4. Biological role of RNA silencing
5. Applications of RNA silencing
1. History
RNA σίγηση, ένας μηχανισμόςρύθμισης και άμυνας
Applications of RNA silencing
• Functional analysis of genes
• Sequence specific suppression of unwanted genes
• Generation of plants resistant to viruses
Functional analysis of gene by reverse genetics
1. Identification of gene sequence
2. Reverse genetics:-find copy number/possibly locus (Arabidopsis)-charachterise time and space of expression-find gene function a. overexpressionb. suppression (knock-down, knock-out)
RNAi induced at the level of siRNAs
Direct application of siRNA
(a) chemically synthesized(b) enzymatically synthesized
delivery via short hairpin RNAs (shRNA)
Vector-mediated RNAi (DNA or virus)
Strategies of expressing double-stranded RNA
Pol II and Pol III (type III) e.g U6 prom
Promoter spacer/intron
terminator
AAA
alternative: tandem promoters, opposing promoters
Hairpin induced gene silencing of CP gene
RB
35S
(+) CPfrag.
spacer nptII
LB(-)CP frag.
ter
~ ~~ ~ ~Dicer
~ ~~ ~ ~~ ~~ ~ ~
RISC
~ ~~ ~ ~
RNA σίγηση, ένας μηχανισμόςρύθμισης και άμυνας
2. The role of small RNAs in gene regulation
3. Mechanisms of RNA interference
4. Biological role of RNA silencing
5. Applications of RNA silencing
6. miRNAs
1. History and definitions
MicroRNAs
• short (20-25nt) RNA molecules• translated as precursor RNA
molecule• post transcriptional gene regulation• target mRNA for cleavage or
translational repression• very abundant class of genes
The processing pathway of siRNA & miRNA
from Dykxhoorn et al., (2003) Nature Reviews 4, 457-466.
Mechanism of miRNA suppression of gene expression
• Transcription• mRNA degradation• Translational repression
– 1 Initiation– 2 Elongation– 3 Termination– 4 Release
• Co-translational degradation of the nascent peptide
Cullen Nature Immunology 7:563 2006
Published by AAASJ. Liu et al., Science 305, 1437 -1441 (2004)
Identification of Argonaute 2 as Slicer in humans
miRNA• The miRNA are endogenous small RNA guides that
repress the expression of target genes.• Differ from siRNA in biogenesis not in functions,
although mechanisms can be different. mRNA cleavage when complementarity is extensive, repress translation when not.
• lin-4 mutant worms had defects in timing of cell division. Encodes a small RNA that binds to and silenced lin-14 message.
• Lin-14 mRNA levels do not decline, but that may not always be the case.
• let-7 also found in other species.
miRNA
• Many miRNA are embedded in introns of protein encoding genes and are transcribed together with host genes.
• miRNA can be expressed in developmentally tissue specific fashion but may not be expressed in tissues where putative target sequences are.
Du, T. et al. Development 2005;132:4645-4652
The structure of human pri-miRNAs
Useful plant miRNA properties
• (at least some) miRNAs are conserved between species
• most precursor sequences are not conserved
• precursor structure is somewhat conserved
• mature miRNA always cut from 1 arm of RNA helix in precursor
• plant precursors are more complex
Plasterk Cell 124:877, 2006
PLoS, 39 (3): 404-418
Summary of plant miRNA/target recognition
1. Perfect match for positions 2-12
2. No more than 2 consecutive mismatches at 3’
3. Free energy at least 72% that of perfect match
Esquela-Kerscher et al. Nature Reviews Cancer 6, 259–269 (April 2006) | doi:10.1038/nrc1840
MicroRNAs can function as tumour suppressors and oncogenes
And yet more roles for small RNAs…
RNA σίγηση, ένας μηχανισμόςρύθμισης και άμυνας
2. The role of small RNAs in gene regulation
3. Mechanisms of RNA interference
4. Biological role of RNA silencing
5. Applications of RNA silencing
6. miRNAs
1. History and definitions
7. RNA-mediated epigenetic changes
Herve Vaucheret Genes Dev. 2006; 20: 759-771
Detection of methylation in DNADetection of methylation in DNA
HpaII CCGG
MspII CCGG
CMG
CMNG
HaeIII GGCC CMNN
CCMGG
CMCGG
GGCMC
Restrictionenzyme
cut no cut detection of
or bisulphite sequencing in combination with PCR
Discovery of DNA-dependent Pol IV in plantsDiscovery of DNA-dependent Pol IV in plants
Nature Reviews Genetics 4; 29-38 (2003); doi:10.1038/nrg982EXPLORING PLANT GENOMES BY RNA-INDUCED GENE SILENCING